High fragmentation characterizes tumour-derived circulating DNA.

SysDiag UMR3145-CNRS, National Centre of the Scientific Research/BIO-RAD, Montpellier, France.

Abstract

BACKGROUND:

Circulating DNA (ctDNA) is acknowledged as a potential diagnostic tool for various cancers including colorectal cancer, especially when considering the detection of mutations. Certainly due to lack of normalization of the experimental conditions, previous reports present many discrepancies and contradictory data on the analysis of the concentration of total ctDNA and on the proportion of tumour-derived ctDNA fragments.

METHODOLOGY:

In order to rigorously analyse ctDNA, we thoroughly investigated ctDNA size distribution. We used a highly specific Q-PCR assay and athymic nude mice xenografted with SW620 or HT29 human colon cancer cells, and we correlated our results by examining plasma from metastatic CRC patients.

(A): Athymic nude mice were xenografted with HT29 cells and sacrificed at various time points and thus with tumours of increasing weight (137, 189, 310, 380, 400, 1019, 1080 mg from Mo1 to Mo7, respectively). CtDNA concentration (ng/ml plasma, left y axis) (dashed line) was assessed by Q-PCR using a primer set that amplifies a 133 bp sequence from human ACTB (). Tumour-derived ctDNA fragmentation (full line) was estimated by calculating the DII (right y axis). Here, the DII corresponds to the ratio of the concentration found when targeting a 290 bp sequence of the human ACTB gene to the concentration found when targeting a 133 bp sequence located within the former 290 bp region (). (B): Tumour ctDNA was quantified by Q-PCR with human KRAS 73 (empty bars), KRAS 145 (hatched bars) and KRAS 300 (full bars) primer sets () in plasma samples from SW620 xenografted mice. Mo8, Mo9 and Mo10 correspond to three non-xenografted nude mice. CtDNA fragmentation was estimated by calculating the 300/145 and 300/73 DII. DII was calculated as the ratio of the ctDNA concentrations obtained from the amplification of a short 73 bp, a medium 145 bp and a long 300 bp target sequence from the human KRAS gene (). Bars represent ctDNA concentration expressed as ng/ml of plasma (left y axis) and curve represents tumour weight (mg, right y axis). Data represent the mean values of the ctDNA concentrations obtained in duplicates. NC, not calculated. No ctDNA was detected with the KRAS 300 bp primers in Mo11 and Mo12 plasmas.

CtDNA fragment size profiles determined by Q-PCR for amplicons of different length in xenografted mice.

Evaluation of tumour and non-tumour ctDNA concentration in plasma samples from mice xenografted with SW620 cells and of control ctDNA in non-xenografted nude mice was determined using a multi-integrated Q-PCR system for detecting amplicons of increasing length in intron 2 of human wild type KRAS () or in intron 7 of mouse wild type KRAS (). Tumour ctDNA, non-tumour ctDNA and control ctDNA concentrations are expressed as ng/ml of plasma (A) or as % of the highest value of each concentration set (B). Each point represents the mean of three pools of plasma from three mice with 300–550 mg tumours. The relative percentage of tumour and non-tumour ctDNA from xenografted mice for each amplicon length is presented in (C): Tumour (black bars) and non-tumour (hatched bars) ctDNA mean concentrations are expressed as ng/ml of plasma; the bar height is the sum of tumour and non-tumour ctDNA concentrations (estimated as the total ctDNA concentration). For clarity human and mouse amplified targeted sequences of close size were grouped together: 60 (60 and 63), 100 (101 and 95), 150 (145 and 150), 200 (185 and 196), 250 (249 and 256), 350 (354 and 357) and 400 (409 and 382) bp. Fractional fragment size distribution of ctDNA amount from tumour and non-tumour ctDNA in xenografted mice and control ctDNA in non-xenografted mice (D). The ctDNA amount was arbitrarily estimated for the 60–100, 100–150, 150–400 and >400 bp fragment size ranges. The estimated amount of ctDNA (ng) in one ml of plasma in each range of ctDNA size was calculated as described in section.

Comparison of ctDNA size profile upon amplicon length in healthy individuals (HHP) and CRC patients (A). CtDNA amount was calculated as described in section. The corresponding concentration values for each CRC and HHP plasma sample are presented in . HHP is the mean from plasmas of two healthy individuals (HHP1 and HHP2) and of a pool of 7 healthy individuals (HHP3). The estimated amount of ctDNA in each range of ctDNA size was calculated as described in section. HHP and CRC ctDNA concentration are expressed as % of total ctDNA value of each profile (B). CtDNA concentration was determined using a multi-integrated Q-PCR system for detecting amplicons of increasing length in intron 2 of human wild type KRAS () as performed in .

Comparison of the fractional fragment size distribution of ctDNA from clinical samples and from the animal model.

CRC and healthy patients (A) and tumour and non-tumour ctDNA in xenografted mice and control ctDNA in non-xenografted mice (B). The estimated amount of each ctDNA fraction was expressed as the percentage to the total ctDNA amount estimated as the sum of the ctDNA amount of the four size fractions. Data were calculated from the experiments presented in , (mouse plasma samples) and (human plasma samples).

Discrimination between healthy and CRC subjects by comparing DII values.

DII ratio between HHP and CRC patients (A) and between control non-xenografted and SW620 xenografted mouse plasma (B) were determined from DII values represented in and , respectively. DII of plasma from each CRC patient was compared with the mean of healthy individuals (HHP, n = 9). Mouse DII ratios are determined from the mean of 3 pools from 3 mice (n = 9). The DII was estimated by the ratio of the concentration of amplicons of increasing size and the concentration to the 60 bp amplicons.

Comparison of the DII values (A) from genomic DNA, and ctDNA from mice plasma (non-xenografted and xenografted) and (B) from human plasmas (healthy and CRC). The DII was estimated by the ratio of the concentration obtained by targeting a 300 bp sequence and a 60 bp sequence in a KRAS region.